Gene delivery for therapy or other purposes is of course well-known, particularly for the treatment of diseases such as cystic fibrosis and certain cancers. The term refers to the delivery into a cell of a gene or part of a gene to correct some deficiency. In the present specification the term is used also to refer to any introduction of nucleic acid material into target cells, and includes gene vaccination and the in vitro production of commercially-useful proteins in so-called cell factories.
Cell delivery systems fall into three broad classes, namely those that involve direct injection of naked DNA, those that make use of viruses or alternated viruses and those that make use of non-viral delivery agents. Each has its advantages and disadvantages. Although viruses as delivery agents have the advantages of high efficiency and high cell selectivity, they have the disadvantages of toxicity, production of inflammatory responses and difficulty in dealing with large DNA fragments. The present invention, in making use of lipids, can overcome these problems.
Non-viral gene delivery systems are based on the compaction of genetic material into nanometric particles by electrostatic interaction between the negatively charged phosphate backbone of DNA and cationic lipids, peptides or other polymers (Erbacher, P. et al, Gene Therapy, 1999, 6, 138-145). Various mechanisms for the action of these species have been suggested. An early suggestion was that membrane fusion between liposome and cell membrane occurs. More recently, endocytosis of intact complexes has been proposed. Complexes formed between the nucleic acid and the lipid become attached to the cell surface, and then enter by endocytosis. They then remain localised within a vesicle or endosome for some time and the nucleic acid component is then released into the cytoplasm. Migration of the nucleic acid into the nucleus may then occur some time later, where a gene encoded by the nucleic acid may be expressed. Gene expression in the nucleic involves decoding DNA into RNA and then the protein.
The use of lipids, rather than viruses, for this purpose can result in lower toxicity, reduced cost, reasonably efficient targeting, and the ability to deal with large fragments of nucleic acid material. Unfortunately, lower transfection efficiencies have been noted.
Known complexes include LD complexes comprising DNA condensed with cationic lipids. The DNA is protected from degradation by endogenous nucleases or immune reaction, as well as lysosomal degradation, and can be internalised by endocytosis. Complexes of these cytofectins with DNA need to have an overall positive charge in order to transfect cells as well as avoid aggregation. However, these complexes cannot target specific cells and can have undesirable interactions with the plethora of anionic particles found in extracellular fluids of higher organisms.
Complexes can also be formed between targeting peptides that target cell surface receptors, such as integrin-targeting (I-) peptides, and DNA. When such complexes comprise I-peptides they are called ID complexes. These complexes can be designed to be cell specific and have transfection efficiencies similar to LD complexes. However ID complexes are susceptible to endosomal degradation due to their nature.
A preferred complex is a “LID complex”. As used herein, the term “LID complex” represents a complex comprising a lipid, an integrin- (or other receptor-) binding peptide and DNA. These complexes combine the advantages of both LD and ID complexes. LID complexes achieve transfection via an integrin-mediated pathway; they do not necessarily need to have an overall positive charge so undesirable serum interaction can be reduced. The lipid component shields both DNA and, to a degree, the peptide component from degradation, endosomal or otherwise. The peptide component can be designed to be more or less integrin-specific thus conferring a given degree of cell specificity to the LID complex itself. Specificity results from the targeting of the integrin, and transfection efficiencies comparable to some adenoviral vectors can be achieved (Hart et al., Hum. Gene Ther. 9, 1037-47, 1998; Harbottle et al. Hum. Gene. Ther. 9, 575-85, 1998; and Jenkins et al. Gene Therapy 7. 393-400, 2000, the disclosures of which are incorporated herein by reference).
The components of a LID complex associate electrostatically to form a Lipid/Peptide vector complex (Hart et al., Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector, Hum Gene Ther 1998, 9, 575-85; Meng et al., Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system, J Gene Med 2004, 6, 210-21; Parkes et al., High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system, J Gene Med 2002 4, 292-9). A LID complex is thus a lipopolyplex type of vector. Lipid/peptide vectors transfect a range of cell lines and primary cell cultures with high efficiency epithelial cells (40% efficiency), vascular smooth muscle cells (50% efficiency), endothelial cells (30% efficiency) and haematopoietic cells (10% efficiency), and low toxicity. Furthermore, in vivo transfection of bronchial epithelium of mouse has been demonstrated (Jenkins et al., Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo, Gene Therapy 2003, 10, 1026-34), rat lung (Jenkins et al., An integrin-targeted non-viral vector for pulmonary gene therapy, Gene Therapy 2000, 7, 393-400) and pig lung (Cunningham et al., Evaluation of a porcine model for pulmonary gene transfer using a novel synthetic vector, J Gene Med 2002, 4, 438-46) and with efficiency comparable to that of an adenoviral vector (Jenkins et al., 2000, as above).
An I-peptide for use in such LID complexes must have two functionalities: a “head group” containing an integrin recognition sequence and a “tail” that can bind DNA non-covalently. These two components must also be covalently linked in a way that does not interfere with their individual functions. This is the role of the spacer.
A known I-peptide is peptide 6, in which glycine-alanine is the spacer:

Turning now to the lipid component of the LID complexes, cationic lipids for such a use were developed by Felgner in the late 1980s, and reported in Proc. Natl. Acad. Sci. USA 84, 7413-7417. 1987. A patent to Felgner et al. that may be referred to is U.S. Pat. No. 5,264,618. The disclosure of each of these documents is incorporated herein by reference. Felgner developed the now commercially-available cationic liposome known by the trade mark “Lipofectin” which consists of the cytofectin, DOTMA and the neutral lipid DOPE in a 1:1 ratio. Various other cationic liposome formulations have since been devised, most of which combine a synthetic cationic cytofectin and a neutral lipid. However, cationic vector systems vary enormously in their transfection efficiencies in the presence of serum, which clearly impacts on their potential uses for in vivo gene therapy.